外泌体负载的KV11通过VDAC1和自噬机制对角膜新生血管的抑制作用  

Inhibitory effect of exosome-loaded KV11 on corneal neovascularization via VDAC1 and autophagy

作　　者：陈文倩 杜玮 于文贞[1] Chen Wenqian;Du Wei;Yu Wenzhen(Department of Ophthalmology,Peking University People's Hospital,Eye Diseases and Optometry Institute,Beijing Key Laboratory of Diagnosis and Therapy of Retinal and Choroid Diseases,College of Optometry,Peking University Health Science Center,Beijing 100044,China)

机构地区：[1]北京大学人民医院眼科,北京大学人民医院眼视光中心,北京大学人民医院眼病与视光医学研究所,视网膜脉络膜疾病诊治研究北京市重点实验室,北京大学医学部眼视光学院,北京100044

出　　处：《中华实验眼科杂志》2024年第2期108-116,共9页Chinese Journal Of Experimental Ophthalmology

基　　金：北京市自然科学基金(京津冀基础研究合作专项) (J190005)。

摘　　要：目的探讨外泌体(EXO)负载的抗新生血管短肽KV11在角膜新生血管(CNV)中的作用及其机制。方法利用EXO锚定肽CP05将KV11结合到血管内皮来源的EXO膜表面,形成EXO-KV11。通过Apogee纳米流式分析EXO负载KV11的效率和最佳浓度比。选取8周龄SPF级健康雄性SD大鼠100只,其中10只大鼠不做任何处理,为正常对照组;其余大鼠在建立碱烧伤诱导CNV大鼠模型后,采用随机数字表法随机将大鼠分为EXO-KV11组、KV11组和生理盐水组,每组各30只。从碱烧伤后第1天开始每隔1天,各组分别结膜下注射100μl EXO-KV11(25μg)、KV11(25μg)或生理盐水。观察第1、4、7、14天时CNV的生成情况;采用荧光素心室灌注、角膜血管造影法计算CNV面积;采用苏木精-伊红染色法观察各组CNV管腔数量;采用免疫组织化学法检测角膜组织中CD31的表达分布;采用Western blot法检测电压依赖性阴离子通道1(VDAC1)和内质网应激、自噬及凋亡相关蛋白的表达水平。结果Apogee流式分析确定KV11与EXO最佳浓度比为4∶1,EXO负载KV11效率高达87.5%。碱烧伤后7 d和14 d,各组CNV面积总体比较差异均有统计学意义(F=4.613、15.590,均P<0.05),其中碱烧伤后7 d,EXO-KV11组CNV面积小于KV11组和生理盐水组;碱烧伤后14 d,EXO-KV11组和KV11组CNV面积均小于生理盐水组,EXO-KV11组CNV面积小于KV11组,差异均有统计学意义(均P<0.05)。角膜荧光铺片定量分析结果显示,生理盐水组、KV11组和EXO-KV11组CNV相对荧光面积分别为(8.3±1.7)%、(5.2±1.6)%和(3.4±0.7)%,总体比较差异有统计学意义(F=11.735,P<0.01),其中KV11组和生理盐水组CNV相对荧光面积均大于EXO-KV11组,生理盐水组CNV相对荧光面积大于KV11组,差异均有统计学意义(均P<0.05)。碱烧伤后14 d,生理盐水组基质内可见大量新生血管管腔;KV11组基质内新生血管管腔数量较生理盐水组少;EXO-KV11组角膜结构趋于正常,少见新生血管管腔。生理盐水组角膜基�Objective To investigate the effects and mechanism of exosome(EXO)-loaded kringle V11(KV11)delivery on corneal neovascularization(CNV).Methods KV11 was bound to the surface of endothelial cell-derived exosomes by using CP05,an EXO-targeting anchoring peptide,to produce EXO-KV11.The binding efficiency and optimal concentration ratio were determined using the Apogee flow system.A total of 1008-week-old healthy male SPF grade SD rats were selected,10 of which were randomly selected as a normal control group without any treatment.The CNV model was established by alkali burn in the other 90 rats,which were randomly divided into three groups,EXO-KV11 group,KV11 group,and normal saline group by the random number table method,with 30 rats in each group.Each group was injected subconjunctivally with 100μl of EXO-KV11(25μg),KV11(25μg),or normal saline every other day from the first day after the alkali burn,respectively.The CNV of rats was observed on days 1,4,7,and 14 after alkali burn.The CNV area was calculated by ventricular perfusion with fluorescein isothiocyanate-dextran(FITC-dextran)and corneal angiography.The amount of CNV lumen was observed by hematoxylin and eosin staining.The distribution of CD31 in rat corneas was determined by immunohistochemical method.The expression levels of voltage-dependent anion channel 1(VDAC1),endoplasmic reticulum stress,autophagy and apoptosis-associated proteins were detected by Western blot.This study was approved by the Animal Ethics Committee of Peking University People's Hospital(No.20210019).All animal procedures complied with the regulations of the Vision and Ophthalmology Association and the Animal Protection and Use Committee of Peking University.Results The optimal concentration ratio of KV11 to EXO was 4∶1 and the binding affinity reached up to 87.5%by Apogee flow cytometers.On days 7 and 14 after alkali burn,there were significant differences in CNV area among the four groups(F=4.613,15.590;both at P<0.05).On day 7 after alkali burn,the CNV area was smaller in EXO-K

关 键 词：角膜新生血管 自噬 外泌体 纤溶酶原kringle 5 电压依赖性阴离子通道1 

分 类 号：R772.2[医药卫生—眼科]